Spot-on pesticide composition

ABSTRACT

A spot-on pesticide composition for animals, specifically mammals, including dogs and cats, which composition comprises a combination of active components, including fipronil and a pyrethroid, and optionally an insect growth regulator, in doses and proportions which are parasiticidally effective against a variety of insects and pests, and in a formulation which is convenient for local application to the animal&#39;s skin, preferably localized over a small surface area.

RELATED APPLICATIONS

This patent application relates to and claims the priority of U.S.Provisional Ser. No. 61/297,154, which was filed Jan. 21, 2010 and U.S.Provisional Patent Application No. 61/244,788, which was filed Sep. 22,2009, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a spot-on or pour-on pesticidecomposition comprising fipronil and a pyrethroid, which combination isuseful in the treatment or prevention of insect, parasite, or tickinfestations in animals, specifically mammals, including dogs and cats.The present invention further relates to a spot-on or pour-on pesticidecomposition comprising fipronil, a pyrethroid, and an insect growthregulator, which combination is also useful in the treatment andprevention of insect, parasite, or tick infestations in animals,specifically mammals. The present invention also relates to a method forthe localized cutaneous application of a pesticide compositioncomprising fipronil and a pyrethroid, and additionally an insect growthregulator.

BACKGROUND OF THE INVENTION

Traditional products for the treatment or prevention of insect orparasite infestation of animals include shampoo treatments, insecticidalcollars, orally ingested treatments, compositions designed to treat ananimal's environment, spot-on treatments, and the like. Differenttreatment forms offer unique benefits and drawbacks; however, themajority offer substantial disadvantages. For instance, shampootreatments require that the treatment be applied over the entire surfaceof the animal and subsequently rinsed off, which is typically unpleasantfor both the animal and the owner and only provides a short-term,transient treatment. Insecticidal collars require the animal tophysically wear the collar for a period of time often lasting severalmonths, which is uncomfortable and burdensome to the animal. Additional,treatments administered orally tend to increase the possibility of sideeffect and are more difficult to administer to the animal.Alternatively, treatment of the animal's surroundings and habitat isoften undesirable due to the fact that the treatment may causediscoloration of furniture, carpet, bedding, etc., and may also produceunpleasant odors. Thus, it is desirable to have a spot-on treatment thatcan be applied to the animal in smaller portions, while maintainingtreatment efficacy across the entire body surface of the animal.

Spot-on compositions that have been previously developed incorporate amultitude of pesticide agents. Common agents include arylpyrazolederivatives, insect growth regulators, pyrethroids, nodulisporic acidderivatives, neonicotinoids, formamides, avermectins, and the like. Allof the compounds listed herein have different mechanisms of action, andaccordingly treat and prevent infestation in different manners.Consequently, the various compounds also have a variety of differentadverse effects associated with treatment. The various agents may becombined in a variety of concentrations. Generally, higherconcentrations of the active components result in higher pest killrates, and more successful treatments; however, the use of higherconcentrations of the active components are more expensive to make andresult in a greater likelihood that the animal will suffer adverseeffects from treatment. Adverse effects of treatments include skindiscoloration, local hair loss, itching, redness, excessive salivation,and in certain cases, neurotoxicity.

The spot-on treatments known within the art generally have a prolongedperiod of action before the active ingredient(s) effectively eliminatesthe target pest. For instance, insect growth regulators (i.e. juvenilehormone mimetics) exterminate target pests by effectively inhibiting thedevelopment of immature pests, so that they are not able to reproduce.Even though the insect growth regulators are effective in ultimatelycontrolling the pest infestation, additional time is required to killall pests, which leads to additional time in which the animal host, aswell as all other animals and humans, must suffer the effects of theinfestation. Even quick-acting agents, such as the arylpyrazolederivative known as fipronil, which causes hyperexcitation of the pestleading to its death, have a prolonged onset of action. Generally, itmay take multiple hours for quick-acting agents to provide symptomaticrelief to the host animal.

Therefore, given the limitations of the prior art, it would be desirableto have a spot-on pesticide treatment that utilizes low concentrationsof known chemicals so as to minimize the risk of adverse effects, has ahigh pest kill rate, and has an improved kill rate, preferably withinthe first hour of treatment.

SUMMARY OF THE INVENTION

The invention relates to novel spot-on compositions for treating andpreventing insect or tick infestation, as well as a method of killingpests comprising applying the compositions to a host animal,specifically a mammal. The spot-on insecticidal composition of thecurrent invention comprises low concentrations of the active componentsfipronil and a pyrethroid, and may additionally include an insect growthregulator, in which the active agents are each present in thecomposition in concentrations generally less than 20% of the totalweight of spot-on composition (w/w). Such low concentrations minimizethe risk of adverse effects. It has further been discovered that thesenovel combinations of active components have a higher and faster killrate of pests (e.g. fleas and ticks) than treatment with fipronil and/oran insect growth regulator alone or without a pyrethroid. Thecompositions of the present invention further comprise an organicsolvent and may optionally include an antioxidant.

One embodiment of the current invention relates to a spot-on compositioncomprising between about 1% and about 20% (w/w) fipronil, between about1% and about 20% (w/w) pyrethroid, between about 65% to about 85% (w/w)organic solvent, and between about 2% to about 10% (w/w) antioxidant.More specifically, this particular embodiment of the spot-on compositionincludes between about 5% and about 15% (w/w) fipronil, between about 2%and about 10% (w/w) pyrethroid, between about 70% to about 80% (w/w)organic solvent, and between about 3% and about 8% (w/w) antioxidant.Most specifically, the first embodiment of the present inventionincludes between about 8% and about 11% (w/w) fipronil, between about 4%and about 6% (w/w) pyrethroid, between about 75% and about 80% (w/w)organic solvent, and between about 4% and about 6% (w/w) antioxidant.

A second embodiment of the current invention relates to a spot-oncomposition comprising between about 1% and about 20% (w/w) fipronil,between about 1% and about 20% (w/w) pyrethroid, between about 1% andabout 20% (w/w) insect growth regulator, between about 55% and about 80%(w/w) organic solvent, and between about 2% and about 10% (w/w)antioxidant. More specifically, this embodiment of present compositionincludes between about 5% and about 15% (w/w) fipronil, between about 2%and about 10% (w/w) pyrethroid, between about 4% and about 15% (w/w)insect growth regulator, between about 60% and about 75% (w/w) organicsolvent, and between about 3% and about 8% (w/w) antioxidant. Mostspecifically, the spot-on composition of the present invention includesbetween about 8% and about 11% (w/w) fipronil, between about 4% andabout 6% (w/w) pyrethroid, between about 7% and about 11% (w/w) insectgrowth regulator, between about 65% and about 70% (w/w) organic solvent,and between about 4% and about 6% (w/w) antioxidant.

A third embodiment of the current invention relates to a spot-oncomposition comprising between about 1% and about 20% (w/w) fipronil,between about 1% and about 20% (w/w) pyrethroid, and between about 60%to about 85% (w/w) organic solvent. More specifically, this embodimentof the spot-on composition includes between about 5% and about 15% (w/w)fipronil, between about 8% and about 18% (w/w) pyrethroid, and betweenabout 65% and about 80% (w/w) organic solvent. Most specifically, thisembodiment of the of the present invention includes between about 8% andabout 11% (w/w) fipronil, about 14% and about 16% (w/w) pyrethroid, andbetween about 70% and about 75% (w/w) organic solvent.

A fourth embodiment of the spot-on composition includes between about 1%and about 20% (w/w) fipronil, between about 1% and about 20% (w/w)pyrethroid, between about 1% and about 20% (w/w) insect growthregulator, and between about 55% and about 75% (w/w) organic solvent.More specifically, this embodiment of present composition includesbetween about 5% and about 15% (w/w) fipronil, between about 8% andabout 18% (w/w) pyrethroid, between about 5% and about 16% (w/w) insectgrowth regulator and between about 60% and about 70% (w/w) organicsolvent. Most specifically, this embodiment of the spot-on compositionincludes between about 8% and about 11% (w/w) fipronil, between about14% and about 16% (w/w) pyrethroid, between about 11% and about 14%(w/w) insect growth regulator, and between about 60% and about 65% (w/w)organic solvent.

A fifth embodiment of the spot-on composition includes between about 1%and about 20% (w/w) fipronil, between about 1% and about 20% (w/w)pyrethroid, between about 1% and about 20% (w/w) insect growthregulator, between about 65% and about 85% (w/w) organic solvent, andbetween about 2% and about 10% (w/w) antioxidant. More specifically,this embodiment of present composition includes between about 5% andabout 15% (w/w) fipronil, between about 2% and about 10% (w/w)pyrethroid, between about 2% and about 10% (w/w) insect growthregulator, between about 70% and about 85% (w/w) organic solvent, andbetween about 3% and about 8% (w/w) antioxidant. Most specifically, thespot-on composition of the present invention includes between about 8%and about 11% (w/w) fipronil, between about 4% and about 6% (w/w)pyrethroid, between about 3% and about 6% (w/w) insect growth regulator,between about 75% and about 80% (w/w) organic solvent, and between about4% and about 6% (w/w) antioxidant.

A sixth embodiment of the spot-on composition includes between about 1%and about 20% (w/w) fipronil, between about 1% and about 20% (w/w)pyrethroid, between about 1% and about 20% (w/w) insect growthregulator, and between about 55% and about 80% (w/w) organic solvent.More specifically, this embodiment of present composition includesbetween about 5% and about 15% (w/w) fipronil, between about 8% andabout 18% (w/w) pyrethroid, between about 2% and about 10% (w/w) insectgrowth regulator, and between about 60% and about 75% (w/w) organicsolvent. Most specifically, this embodiment of the spot-on compositionincludes between about 8% and about 11% (w/w) fipronil, between about14% and about 16% (w/w) pyrethroid, between about 3% and about 6% (w/w)insect growth regulator, and between about 65% and about 70% (w/w)organic solvent.

In addition, the present invention further provides a method ofeliminating and preventing pest infestations on an animal, specificallya dog, the method comprising administering a localized cutaneousapplication of a spot-on composition of the present invention betweenthe two shoulders of the animal in a volume sufficient to deliver a doseof the active components ranging from about 0.5 mg/kg to about 10 mg/kgof animal body weight.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs at the time of filing. Ifspecifically defined, then the definition provided herein takesprecedent over any dictionary or extrinsic definition. Further, unlessotherwise required by context, singular terms shall include pluralities,and plural terms shall include the singular. Herein, the use of “or”means “and/or” unless stated otherwise. All patents and publicationsreferred to herein are incorporated by reference.

DETAILED DESCRIPTION OF THE INVENTION

The compositions provided herein are spot-on pesticide compositions thatutilize combinations of certain active compounds to treat insect,parasite, or tick infestation of animals, specifically mammals(preferably dogs and cats), and also prevent future infestations byprolonged treatment efficacy that can last up to three months. As such,the compositions exterminate existing pests, and prevent those peststhat survive from developing and reproducing. The compositions halt thegrowth cycle and prevent pests from laying additional eggs. Thecompositions of the current invention are useful in the treatment ofmany pests, especially fleas and ticks found on domesticated animals.The compositions include low concentrations of fipronil and apyrethroid, and may further comprise an insect growth-regulatingcompound. In addition, the present invention is based in part on thefinding that treatment of a host animal with compositions comprising acombination of fipronil and a pyrethroid results in dramatically higherkill rates within the first twenty-four hours of treatment than doestreatment with fipronil and an insect growth regulator, alone orcombined, without the addition of a pyrethroid.

The spot-on compositions of the present invention include fipronil. Thefipronil compound is a phenylpyrazole acaricide with efficacy against abroad spectrum of tick species and was first disclosed in U.S. Pat. No.5,232,940. Fipronil achieves its efficacy by disrupting the centralnervous system by blocking the passage of chloride ions through the GABAreceptor and glutamate-gated chloride channels (GluCl), components ofthe central nervous system. This disruption causes hyperexcitation ofcontaminated nerves and muscles, which results in eventual death. Thecompound is a slow-acting acaricide, and as such, can be used to targetnot only the host, but also other ticks in which the host comes incontact. Fipronil is also known as5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(1-R,S) (trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile,5-amino-1-(2,6-dichloro-α,α,α-trifluoro-p-tolyl)-4-[(trifluoromethyl)sulfinyl]pyrazole-3-carbonitrile, and fluocyanobenpyrazole [CAS No.120068-37-3]. Fipronil is generally available as either a liquid orsolid crystalline substance or powder. Fipronil typically comprisesbetween about 1% and about 20% (w/w) of the total weight of the spot-oncomposition. In some embodiments, fipronil comprises about 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, or 1% (w/w) of the spot-on composition. For example, the amount offipronil present in the spot-on composition may range from between about5% to about 15% (w/w) of the total composition, and preferably rangesfrom between about 7% and about 12% (w/w). Most preferably, the amountof fipronil present in the spot-on composition may range from betweenabout 8% and about 11% (w/w) of the total composition. In an exemplaryembodiment, the amount of fipronil present in the composition is 9.8%(w/w) of the total composition. The chemical structure for fipronil isshown below.

The spot-on compositions of the present invention also include apyrethroid compound. Generally, pyrethroids are a class of syntheticinsecticides that are related to the naturally-occurring pyrethrins.Pyrethroids tend to be more effective than the natural pyrethrins, andless toxic to mammals. Pyrethroids are axonic poisons that work bykeeping the sodium channels open in the neuronal membranes. The sodiumchannel consists of a membrane protein with a hydrophilic interior whichpermits sodium ions to enter and exit the membrane. When the sodiumchannels are kept open, the influx of sodium ions results inhyperexcitation, and the pest becomes paralyzed. The pyrethroidtypically comprises between about 1% and about 20% (w/w) of the totalweight of the spot-on composition. In some embodiments, the pyrethroidcomprises about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%,9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (w/w) of the spot-on composition.For example, the amount of a pyrethroid present in the spot-oncomposition may range from between about 1% to about 18% (w/w) of thetotal composition, and preferably ranges from about 3% to about 16%(w/w). In another embodiment, the amount of a pyrethroid present in thespot-on composition may range from about 1% to about 10% (w/w) of thetotal composition weight. In a further embodiment, the amount of apyrethroid present in the spot-on composition may range from about 4% toabout 6% (w/w) of the total composition. In still another embodiment,the amount of a pyrethroid present in the spot-on composition may rangefrom between about 10% to about 16% (w/w) of the total composition. In afurther embodiment, the amount of a pyrethroid present in the spot-oncomposition may range from between about 14% to about 16% (w/w) of thetotal composition. Suitable non-limiting examples of pyrethroids includepermethrin, cypermethrin, cyphenothrin, etofenprox, fenvalerate, andcyfluthrin. Specifically, cyphenothrin is classified as a pyrethroidester insecticide. Cyphenothrin is also known as(RS)-α-cyano-3-phenoxybenzyl(1RS,3RS;1RS,3SR)-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate,d-trans-cyphenothrin, d-cyphenothrin, Gokilaht™,(RS)-α-cyano-3-phenoxybenzyl-(1RS)-cis-trans-2,2-dimethyl-3-(2-methylprop-1-enyl)cyclopropanecarboxylate, (±)-α-cyano-3-phenoxybenzyl(±)-cis-trans-chrysanthemate, and cyano(3-phenoxyphenyl)methyl2,2-dimethyl-3-(2-methyl-1-propenyl)cyclopropanecarboxylate. Certaincommercially available Gokilaht™ products on the market incorporate bothcyphenothrin and pyriproxyfen. It should be noted that the cyphenothrinproduct of the current invention may only incorporate the activecompound pyriproxyfen in those formulations not also containingS-methoprene due to the discovery that the incorporation of bothS-methoprene and pyriproxyfen in a composition decreases efficacy andincreases the likelihood of adverse effects. The chemical structure forcyphenothrin is shown below.

In an exemplary embodiment, the amount of cyphenothrin present in thespot-on composition effective for the treatment of pest infestations indogs is 5.0% (w/w) of the total composition. Etofenprox is also known asethofenprox, ethophenprox,1-ethoxy-4-[2-methyl-1-[[3-(phenoxy)phenyl]methoxy]propan-2-yl]benzene2-(4-Ethoxyphenyl)-2-methylpropyl 3-phenoxybenzyl ether, 3-Phenoxybenzyl2-(4-ethoxyphenyl)-2-methylpropyl ether, C076840,2-(4-ethoxyphenyl)-2-methylpropyl-3-phenoxybenzyl ether,1-(2-(4-Ethoxyphenyl)-2-methylpropoxy) methyl)-3-phenoxy benzene,alpha-((p-Ethoxy-beta,beta-dimethylphenethyl) oxy)-m-phenoxytoluene. TheCAS Registration number for etofenprox is 80844-07-1. Etofenprox isknown to be an effective pyrethroid for the elimination of pests incats. The chemical structure for etofenprox is shown below.

In an exemplary embodiment, the amount of etofenprox present in thespot-on composition effective for the treatment of pest infestations incats is 15.0% (w/w) of the total composition. The spot-on pesticidecomposition of the current invention may additionally include an insectgrowth regulator (IGR). IGRs are not effective in killing pre-existingpests; they prevent reproduction and further infestation. An IGR isgenerally a compound that is capable of disrupting the growth anddevelopment of pest species, so that the pest cannot mature andreproduce. The IGR typically comprises less than about 20% (w/w) of thetotal weight of the spot-on composition. In some embodiments, the IGRcomprises about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or 0% (w/w) of the spot-oncomposition. For example, the amount of IGR present in the spot-oncomposition may range from between 0% to about 20% (w/w) of the totalcomposition weight, and preferably the IGR ranges from between about 2%to about 15% (w/w) of the total composition. In another embodiment, theamount of IGR present in the spot-on composition may range from about 4%to about 12% (w/w) of the total composition. In a further embodiment,the amount of IGR present in the spot-on composition may range fromabout 7% to about 14% (w/w) of the total composition. In still anotherembodiment, the amount of IGR may range from between about 8% to about12% (w/w) of the total composition. In an additional embodiment, theamount of IGR may range from between about 2% to about 9% (w/w) of thetotal composition. In a further embodiment, the amount of IGR may rangefrom between about 3% to about 5% (w/w) of the total composition. IGRsmay include, but are not limited to juvenile hormone mimics, chitinsynthesis inhibitors, and the like. Suitable non-limiting examples ofinsect growth regulators include bistrifluoron, buprofezin,chlorfluazuron, cyromazine, diflubenzuron, flucycloxuron, flufenoxuron,hexaflumuron, lufenuron, novaluron, noviflumuron, penfluoron,teflubenzuron, triflumuron, epofenonane, fenoxycarb, hydroprene,kinoprene, methoprene, pyriproxyfen, triprene, and combinations thereof.In a preferred embodiment, the insect growth regulator is S-methoprene.Generally, methoprene is a racemic mixture of the R- and S-enantiomersof the compound, however, only the S-enantiomer is active as a juvenilehormone analog. A juvenile hormone analog exerts a therapeutic effect bymimicking the natural juvenile hormones found within pests. Juvenilehormone must be absent for a pupa to molt to an adult, so methoprenetreated larvae are unable to successfully develop from pupa to an adultpest. This action breaks the natural life cycle of the pest, preventingit from maturing and reproducing. S-methoprene is also known asisopropyl (2E,4E,7S)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate.S-methoprene is available in a variety of commercial products and isuseful in controlling long-term pest infestation, while other activecomponents are primarily effective in the immediate, short-termelimination of pests. The pest kill time for treatment with S-methoprenewill vary depending on the typical duration of life for the speciesbeing treated. Unlike some other compounds, S-methoprene is generallyconsidered non-toxic to humans, which has led to its use in thetreatment of well cisterns and a number of food items, including meat,milk, mushrooms, peanuts, rice, and cereals. In an exemplary embodiment,the concentration of S-methoprene present in the spot-on compositioneffective for the treatment of pest infestations in animals is 8.8%(w/w).

In an alternative embodiment (i.e. those embodiments of the presentinvention that do not contain S-methoprene), the insect growth regulatormay be the juvenile hormone analog pyriproxyfen, also known as4-phenoxyphenyl 2-(2-pyridyloxy)propyl ether and Nylar™. The chemicalstructure for pyriproxyfen is shown below.

In an exemplary embodiment, the amount of pyriproxyfen present in thespot-on composition effective for the treatment of pest infestations inanimals is 2.0% (w/w) of the total composition.

The spot-on compositions of the present invention, which arenon-aqueous, also comprise an organic solvent. Generally, the organicsolvent is defined as a carbon-containing chemical that is capable ofdissolving a solid, liquid, or a gas. Although one skilled in the artwill appreciate that a wide variety of solvents may be incorporated intothe current invention, the solvents should generally have a dielectricconstant ranging from about 1 to 40, a low boiling point (less than 100°C.), have a density less than the density of water (less than 1.0 at 20°C.), and generally be soluble with water. In addition, the organicsolvent should cause minimal cutaneous irritation when applied to theskin of an animal, including a dog or cat. Suitable examples of organicsolvents include, but are not limited to, acetyltributyl citrate, fattyacid esters such as dimethyl ester, diisobutyl adipate, acetone,acetonitrile, benzyl alcohol, butyl diglycol, dimethylacetamide,dimethylformamide, dipropylene glycol n-butyl ether, ethanol,isopropanol, methanol, ethylene glycol monoethyl ether, ethylene glycolmonomethyl ether, diethylene glycol monoethyl ether,monomethylacetamide, dipropylene glycol monomethyl ether, liquidpolyoxyethylene glycols, propylene glycol, 2-pyrrolidones such asN-methylpyrrolidone, diethylene glycol monoethyl ether, ethylene glycol,diethyl phthalate, ethoxydiglycol, or combinations thereof. In apreferred embodiment, the organic solvent comprises diethylene glycolmonoethyl ether.

In addition, the organic solvent generally comprises between about 55%to about 85% (w/w) of the spot-on composition. In some embodiments, theorganic solvent comprises about 85%, about 80%, about 75%, about 70%,about 65%, about 60%, or about 55% (w/w) of the total composition. Forexample, the amount of organic solvent present in the spot-oncomposition preferably ranges from between about 60% to about 80% (w/w)of the composition. In another embodiment, the amount of organic solventin the spot-on composition ranges from between about 60% to about 75%(w/w) of the total composition. In an additional embodiment, the amountof organic solvent in the spot-on composition ranges from between about70% to 80% (w/w) of the total composition. In still another embodiment,the amount of organic solvent in the spot-on composition ranges frombetween about 60% to 70% (w/w).

The spot-on composition may further include an antioxidant. Anantioxidant can generally be defined as a compound capable of slowing orpreventing the oxidation of other molecules. Oxidation is a chemicalreaction that transfers electrons from the original substance to anoxidizing agent. Oxidation reactions can produce free radicals, whichstart chain reactions that damage cells. Antioxidants terminate thesechain reactions by removing free radical intermediates, and inhibitother oxidation reactions by being oxidized themselves. Within thespot-on composition, the antioxidant acts as a stabilizer, preventingthe various components from degrading by oxidation processes. Inaddition, many of the commercially-available compositions thatincorporate a pyrethroid, including cyphenothrin have reported that theanimals suffer from adverse effects including paraesthesia (a skinsensation that generally comprises feelings of prickling, itching, andtingling). However, it has been shown that inclusion of an antioxidantinto the spot-on composition helps to prevent the undesirable adverseeffects associated with treatment regimens that include cyphenothrin, asdisclosed in U.S. Ser. No. 12/876,122 entitled LIQUID PEST CONTROLFORMULATION, which is commonly owned and is incorporated herein byreference. It should be noted that the spot-on compositions of thepresent invention do not include crystallization inhibitors.

Antioxidants incorporated into the current invention should generally bemiscible with the organic solvents described herein. The antioxidantalso should not cause irritation to the skin of an animal, specificallya dog or cat, when applied to the animal's skin. In addition, theantioxidant may be natural or synthetic. Suitable antioxidants include,but are not limited to, ascorbic acid and its salts, ascorbyl palmitate,ascorbyl stearate, anoxomer, N-acetylcysteine, benzyl isothiocyanate,m-aminobenzoic acid, o-aminobenzoic acid, p-aminobenzoic acid (PABA),butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeicacid, canthaxantin, alpha-carotene, beta-carotene, beta-carotene,beta-apo-carotenoic acid, carnosol, carvacrol, catechins, cetyl gallate,chlorogenic acid, citric acid and its salts, clove extract, coffee beanextract, p-coumaric acid, 3,4-dihydroxybenzoic acid,N,N′-diphenyl-p-phenylenediamine (DPPD), dilauryl thiodipropionate,distearyl thio dipropionate, 2,6-di-tert-butylphenol, dodecyl gallate,edetic acid, ellagic acid, erythorbic acid, sodium erythorbate,esculetin, esculin, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquino line,ethyl gallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA),eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin,epicatechin, epicatechin gallate, epigallocatechin (EGC),epigallocatechin gallate (EGCG), polyphenol epigallocatechin-3-gallate),flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g.,datiscetin, myricetin, daemfero), flavanones, fraxetin, fumaric acid,gallic acid, gentian extract, gluconic acid, glycine, gum guaiacum,hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic acid,hydroxyglutaric acid, hydroquinone, N-hydroxysuccinic acid,hydroxytryrosol, hydroxyurea, rice bran extract, lactic acid and itssalts, lecithin, lecithin citrate; R-alpha-lipoic acid, lutein,lycopene, malic acid, maltol, 5-methoxy tryptamine, methyl gallate,monoglyceride citrate; monoisopropyl citrate; morin,beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate,oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine,phosphoric acid, phosphates, phytic acid, phytylubichromel, pimentoextract, propyl gallate, polyphosphates, quercetin, trans-resveratrol,rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin,sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaricacid, thymol, tocopherols (i.e., alpha-, beta-, gamma- anddelta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma- anddelta-tocotrienols), tyrosol, vanilic acid,2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., Ionox 100),2,4-(tris-3′,5′-bi-tert-butyl-4′-hydroxybenzyl)-mesitylene (i.e., Ionox330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxy butyrophenone,tryptamine, tyramine, uric acid, vitamin K and derivatives, vitamin Q10,wheat germ oil, zeaxanthin, or combinations thereof. One skilled in theart will appreciate that the antioxidants incorporated into thecomposition (including those listed herein) encompass all potential saltand ester forms of the antioxidants in addition to the pure forms of thecompound. Preferably, the antioxidant comprises a vitamin E compoundsuch as tocopherol acetate, tocopherol linoleate, tocopherol nicotinate,tocopherol succinate, ascorbyl tocopherol phosphate, dioleyl tocopherolmethylsilanol, tocophersolan, and tocopherol linoleate/oleate. In anexemplary embodiment, the antioxidant comprises tocopherol nicotinate[CAS No. 43119-47-7].

In addition, the antioxidant typically comprises less than about 10%(w/w) of the total spot-on composition. In some embodiments, theantioxidant comprises about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.1%, or0% (w/w) of the total composition. For example, the amount ofantioxidant present in the spot-on composition may range from between 2%to about 10% (w/w) of the total composition, and preferably theantioxidant ranges from between about 3% to about 6% (w/w) of the totalcomposition. In a further embodiment, the amount of antioxidant presentin the spot-on composition ranges from between about 4% to about 6%(w/w) of the total composition. In an exemplary embodiment, the amountof antioxidant present in the composition is 5.3% (w/w).

The spot-on composition may further include inactive excipients that areadded to the composition as a result of their incorporation into theindividual active components. For instance, the fipronil component ofthe composition may be provided in a 95% solution, meaning that 95% ofthe fipronil component volume is active fipronil compound and theremaining 5% constitutes inactive excipients that are consequentlyintroduced into the composition, as such the pesticide may not be 100%pure concentrate and may be purchased with other constituents. Oneskilled in the art will recognize that the inactive excipients include,but are not limited to binders, fillers, non-effervescent disintegrants,effervescent disintegrants, preservatives, diluents, lubricants, pHmodifiers, stabilizers, and the like. It should, however, be understoodthat the inactive excipients are typically incorporated as a portion ofthe active ingredient components and comprise a small percentage(generally less than 1%) of the total spot-on composition volume,generally not affecting the physical characteristics of the spot-oncomposition.

It should be understood that the active components of the spot-oncomposition may be provided in the form of pure concentrate (100%concentration) or a diluted composition with additional excipients inthe dosage form (i.e. the amount of active ingredient in the compositionis less than or equal to 99.99%, and the remainder consists of inactiveexcipients). One of skill in the art will appreciate that the volume ofactive component added to the spot-on composition will need to beadjusted to account for the dilution and to ensure the end spot-oncomposition comprises the appropriate final concentration of each of theactive components. One of skill in the art will also appreciate that thevarious components of the spot-on composition may be provided in avariety of dosage forms including, but not limited to powder,briquettes, liquid solution or suspension, pellets, emulsion, aerosol,cream, gel, ointment, and the like.

Additionally, the spot-on pesticide composition of the current inventioncan be produced by contacting the various active components of thespot-on composition with one another to produce a spot-on formulationsuitable for application to an animal's skin. It should be understoodthat the current invention encompasses a variety of physicalformulations; however, the spot-on compositions of the current inventionare generally directed to liquid solutions and suspensions. Theformulations of the present invention may be prepared by standardtechniques known in the art. For instance, in one embodiment where thedesired spot-on formulation is a liquid solution, the composition isproduced by bringing the fipronil and pyrethroid components into contactwith a solvent system and then gently heating and stirring thecomponents until dissolved. In a preferred embodiment, the spot-oncomposition comprising fipronil, a pyrethroid, and a solvent system mayfurther be contacted with an antioxidant, and the combination is thenstirred to create a spot-on composition. A person having ordinary skillin the art will appreciate that the various components of the spot-oncomposition may be contacted and mixed with one another in any orderdesired, so long as solution is adequately stirred and mixed.

The physical characteristics of the spot-on composition may varydepending upon the physical characteristics desired. However, thespot-on composition should be capable of application to the skin of ananimal and provide adequate stasis to allow the active components of thespot-on composition to be absorbed by the host animal. Preferably, thespot-on compositions of the present invention have low viscosity.Viscosity is the measurement of flow resistance due to internal frictionwithin the fluid, and is measured in centistokes (cSt). A lower cStmeasurement means the fluid will flow with less resistance, because ofminimal molecular friction within the fluid. The lower the viscosity thefaster the fluid will flow. High viscosity substances are liquids thatare thick and gelatinous in nature with slow flow. Low viscositysubstances exhibit a fast flow with an example being water at roomtemperature (water at 20° C. has a viscosity of about 1 cSt; 1 cSt=1mm²/second). The spot-on compositions of the present invention typicallyhave a viscosity ranging from about 0.01 mm²/second to about 100mm²/second. In a more preferred embodiment, the spot-on composition hasa viscosity ranging from about 1 mm²/second to about 30 mm²/second. In afurther preferred embodiment, the spot-on composition has a viscosityranging from about 4 mm²/second to about 20 mm²/second.

The basic spot-on composition of the present invention includes fipronilat a concentration ranging between about 1% and about 20% (w/w), apyrethroid at a concentration ranging between about 1% and about 20%(w/w), and an organic solvent at a concentration ranging between about55% and about 85% (w/w) of the total spot-on composition. For the basicspot-on composition, it is preferable to use between about 8% to about11% (w/w) fipronil, about 3% to about 16% (w/w) pyrethroid, and about60% to about 80% (w/w) organic solvent, depending on the type of animalto be treated. The basic spot-on composition may also additionallyinclude an IGR at a concentration ranging between 1% and about 20% (w/w)of the composition, preferably at a concentration ranging between about2% to about 12% (w/w) of the total composition. More preferably, the IGRis present in the composition at a concentration ranging between 4% and12% (w/w) of the total composition. In addition, the basic spot-oncomposition may further include an antioxidant at a concentrationranging between 0% and about 10% (w/w) of the total composition,preferably at a concentration ranging between about 4% and about 6%(w/w) of the total composition.

In a preferred embodiment the present invention provides a spot-oncomposition comprising 9.97% (w/w) fipronil, 5.3% (w/w) cyphenothrin,79.43% (w/w) diethylene glycol monoethyl ether, and 5.3% (w/w)tocopherol nicotinate. This spot-on composition can be used to treat anymammal, but it is very desirable for treating dogs.

Another preferred embodiment of the present invention comprises aspot-on composition comprising 9.97% (w/w) fipronil, 5.3% (w/w)cyphenothrin, 9.21% (w/w) S-methoprene, 70.22% (w/w) diethylene glycolmonoethyl ether, and 5.3% (w/w) tocopherol nicotinate. This spot-oncomposition can be used to treat any mammal, but it is very desirablefor treating dogs.

In yet another preferred embodiment the present invention provides aspot-on composition comprising 9.97% (w/w) fipronil, 15.89% (w/w)etofenprox, and 74.14% (w/w) diethylene glycol monoethyl ether. Thisspot-on composition can be used to treat any mammal, but it is verydesirable for treating cats.

In yet another preferred embodiment the present invention provides aspot-on composition comprising 9.97% (w/w) fipronil, 15.89% (w/w)etofenprox, 12.36% (w/w) S-methoprene, and 61.78% (w/w) diethyleneglycol monoethyl ether. This spot-on composition can be used to treatany mammal, but it is very desirable for treating cats.

In yet another preferred embodiment the present invention provides aspot-on composition comprising 9.97% (w/w) fipronil, 5.3% (w/w)cyphenothrin, 4% (w/w) pyriproxyfen, 75.43% (w/w) diethylene glycolmonoethyl ether, and 5.3% (w/w tocopherol nicotinate. This spot-oncomposition can be used to treat any mammal, but it is very desirablefor treating dogs.

In yet another preferred embodiment the present invention provides aspot-on composition comprising 9.97% (w/w) fipronil, 15.89% (w/w)etofenprox, 4.4% (w/w) pyriproxyfen, and 69.74% (w/w diethylene glycolmonoethyl ether. This spot-on composition can be used to treat anymammal, but it is very desirable for treating cats.

In addition, the current invention further embodies a method of killingpest pupae and adults on an animal comprising administering a localizedcutaneous application between the shoulders of the animal, a spot-oncomposition comprising between about 1% to 20% (w/w) fipronil andbetween about 1% to 20% (w/w) pyrethroid. The method of the using thecomposition of the present invention preferably involves the localizedadministration of the basic spot-on composition, which compositionpreferably comprises between 7% (w/w) to about 12% (w/w) fipronil andabout 3% to about 16% (w/w) pyrethroid, and may additionally include anIGR preferably at a concentration range from between about 4% to about12% (w/w) of the total composition.

The compositions and method according to this invention are intended forapplication to animals, in particular dogs and cats, and are generallyapplied by deposition onto the skin (“spot-on” or “pour-on”application). Treatment typically comprises a localized application overa surface area of less than 10 cm², especially of between 5 and 10 cm².Generally, the spot-on composition should be applied to an area wherethe animal cannot lick the application area, as licking of theapplication area may lead to transient adverse effects, such asexcessive salivation. In particular, application is preferred at twopoints and preferably localized between the animal's shoulders. Afterthe spot-on composition has been applied, the composition diffuses, inparticular over the animal's entire body, and then dries withoutcrystallizing or modifying the appearance (in particular absence of anywhitish deposit or dusty appearance) or the feel of the animal's fur.Further, the method of the current invention is directed to applicationof the spot-on composition to the skin of the animal every four weeks toensure continuous treatment and prevention of pest infestation.Typically, the active constituents are applied to the host animaltogether in a single formulation.

In a preferred embodiment of the invention, the method of killing insectand pest pupae and adults on an animal, comprises administering alocalized cutaneous application between the shoulders of an animal, aspot-on composition comprising 9.97% (w/w) fipronil, 5.3% (w/w)cyphenothrin, 79.43% (w/w) diethylene glycol monoethyl ether, and 5. %(w/w) tocopherol nicotinate. This method can be used to treat anyanimal, but it is very desirable for treating dogs.

Another preferred embodiment of the present invention comprises a methodof killing insect and pest pupae and adults on a dog or cat, comprisingadministering a localized cutaneous application between the shoulders ofan animal, a spot-on composition comprising 9.97% (w/w) fipronil, 5.3%(w/w) cyphenothrin, 9.21% (w/w) S-methoprene, 70.22% (w/w) diethyleneglycol monoethyl ether, and 5.3% (w/w) tocopherol nicotinate. Thismethod can be used to treat any animal, but it is very desirable fortreating dogs.

In yet another preferred embodiment the present invention provides amethod of killing insect and pest pupae and adults on an animal,comprising administering a localized cutaneous application between theshoulders of an animal, of a spot-on composition comprising 9.97% (w/w)fipronil, 15.89% (w/w) etofenprox, and 74.14% (w/w) tocopherolnicotinate. This method can be used to treat any animal, but it is verydesirable for treating cats.

In yet another preferred embodiment the present invention provides amethod of killing insect and pest pupae and adults on an animal,comprising administering a localized cutaneous application between theshoulders of an animal, a spot-on composition comprising 9.97% (w/w)fipronil, 15.89% (w/w) etofenprox, 12.36% (w/w) S-methoprene, and 77.67%(w/w) diethylene glycol monoethyl ether. This method can be used totreat any animal, but it is very desirable for treating cats.

In yet another preferred embodiment the present invention provides amethod of killing insect and pest pupae and adults on an animal,comprising administering a localized cutaneous application between theshoulders of an animal, a spot-on composition comprising 9.97% (w/w)fipronil, 5.3% (w/w) cyphenothrin, 4.0% (w/w) pyriproxyfen, 75.43% (w/w)diethylene glycol monoethyl ether, and 5.3% (w/w) tocopherol nicotinate.This method can be used to treat any animal, but it is very desirablefor treating dogs.

In yet another preferred embodiment the present invention provides amethod of killing insect and pest pupae and adults on an animal,comprising administering a localized cutaneous application between theshoulders of an animal, a spot-on composition comprising 9.97% (w/w)fipronil, 15.89% (w/w) etofenprox, 4.4% (w/w) pyriproxyfen, and 85.63%(w/w) diethylene glycol monoethyl ether. This method can be used totreat any animal, but it is very desirable for treating cats.

In an alternative embodiment, the method of killing insects is carriedout such that the spot-on composition is applied in a volume sufficientto deliver a dosage of the active component fipronil ranging from about0.1 mg/kg to about 40 mg/kg of host animal body weight. In a preferredembodiment, the dose of fipronil ranges from about 2 mg/kg to about 20mg/kg of host animal body weight. In a more preferred embodiment, thespot-on composition application comprises a volume sufficient to delivera fipronil dose ranging from about 5 mg/kg to about 15 mg/kg of hostanimal weight.

In another embodiment, the method of killing insects is carried out suchthat the spot-on composition is applied in a volume sufficient todeliver a dosage of the active pyrethroid component ranging from about0.1 mg/kg to about 40 mg/kg of host animal body weight. In a preferredembodiment, the dose of pyrethroid ranges from about 0.5 mg/kg to about20 mg/kg of host animal body weight. In a more preferred embodiment, thespot-on composition application comprises a volume sufficient to delivera pyrethroid dose ranging from about 0.5 mg/kg to about 10 mg/kg of hostanimal body weight.

In a further embodiment, the method of killing insects is carried outsuch that the spot-on composition further comprises an IGR, and isapplied in a volume sufficient to deliver a dosage of the insect growthregulating active component ranging from about 0.1 mg/kg to about 40mg/kg of host animal body weight. In a preferred embodiment, the dose ofinsect growth regulator ranges from about 0.2 mg/kg to about 20 mg/kg ofhost animal body weight. In a more preferred embodiment, the spot-oncomposition application comprises a volume sufficient to deliver aninsect growth regulator dose ranging from about 0.5 mg/kg to about 10mg/kg of host animal body weight.

One of skill in the art will understand that the dosage ranges providedabove are approximate values that may vary within a broad range. Thevariance in dose is due to the fact that, in practice, the spot-oncomposition will be administered in defined doses and volumes to animalswithin a certain range of weights. As a result, the dosage actuallyapplied to the animal may vary by a factor ranging from 0.1 to 10relative to the preferred dose, without imparting any additional riskspertaining to toxicity or decreased efficacy.

Although the components of the composition are effective against a widevariety of pests and parasites, the composition is especially developedfor the treatment of fleas (including the Ctenocephalides species) andticks (the Rhipecephalus, Ixodes, and Trichodectes species).Furthermore, the frequency of application may be varied according to theneeds of the individual animal, as well as the severity of infestation.The treatment of fleas may be repeated as often as once weekly, or maybe reserved for one-time acute treatments of flea infestation orflare-ups. In one embodiment of the current invention, the treatment offleas may be repeated about every four weeks, five weeks, or six weeks.In another embodiment, the spot-on composition is applied to the hostanimal for a one-time treatment of the pest infestation. With regard tothe treatment of ticks, the application schedule for the spot-oncomposition will vary depending on the type of tick being treated. It isgenerally recommended that treatment of paralytic ticks (Ixodes species)occur more frequently than other species. In an embodiment of thecurrent invention, paralytic ticks are treated at a frequency rangingfrom one to four weeks, with treatment every two weeks being preferred.Other genera of ticks generally have a treatment schedule similar totreatment of flea infestation, preferably ranging from approximatelyfour to six weeks. In another embodiment, the spot-on composition isapplied on a one-time basis for the treatment of tick infestation.

Although the invention described herein is susceptible to variousmodifications and alternative iterations, specific embodiments thereofhave been described in greater detail above. It should be understood,however, that the detailed description of the spot-on composition is notintended to limit the invention to the specific embodiments disclosed.Rather, it should be understood that the invention is intended to coverall modifications, equivalents, and alternatives falling within thespirit and scope of the invention as defined by the claim language.

DEFINITIONS

As used herein, the terms “about” and “approximately” designate that avalue is within a statistically meaningful range. Such a range can betypically within 20%, more typically still within 10%, and even moretypically within 5% of a given value or range. The allowable variationencompassed by the terms “about” and “approximately” depends on theparticular system under study and can be readily appreciated by one ofordinary skill in the art.

As used herein, the term “w/w” designates the phrase “by weight and isused to describe the concentration of a particular substance in amixture or solution.

As used herein, the term “mL/kg” designates milliliters of compositionper kilogram of body weight.

As used herein, the term “a.i.” designates active ingredient.

As used herein, the term “treatment” or “treating” of a condition, suchas pest infestation, includes inhibiting an existing condition orarresting its development; or ameliorating or causing regression of thecondition. The term “preventing” or “prevention” of a condition, such asinsect or pest infestation, includes substantially blocking orinhibiting the development or growth of a condition before it starts.Compositions that treat or prevent infestations herein will preferablyexhibit at least 90% efficacy.

As used herein, the term “pesticide” or “pesticidal” refers to an agentor a composition comprising an agent that is capable of preventing,reducing or eliminating pest infestations. Preferred pesticides of thepresent invention include fipronil, cyphenothrin, and etofenprox.

As used herein, the term “insect growth regulator” or “IGR” refers to anagent that is capable of interrupting or inhibiting the life cycle of apest such that the pest never matures into an adult and becomesincapable of reproducing. Preferred IGRs of the present inventioninclude S-methoprene and pyriproxyfen.

As used herein, the term “animal” refers to a mammal, specifically acompanion animal, including but not limited to dogs, cats, rabbits,ferrets, horses, and hamsters.

As used herein, the term “pest” and “insect” refers to any ectoparasite,including but not limited to fleas, ticks, flies, keds, mosquitos, andmites.

The following examples are intended to further illustrate and explainthe present invention. The invention, therefore, should not be limitedto any of the details in these examples.

Example 1 Method of Making a Fipronil/Cyphenothrin Spot-On PesticideComposition for Dogs

Ingredient Amount for 1000 L Fipronil tech. (As 100%) 98 kg (99.7 kg as98.3%) Cyphenothrin (As 100%) 50 kg (53 kg as 94.4%) TocopherolNicotinate 53 kg Diethylene glycol monoethyl ether Up to 1000 L (about794.3 kg)

The diethylene glycol monoethyl ether and the tocopherol nicotinate werecharged to a vessel and heated to a temperature of 50° C. (about 1hour). Once heated, the fipronil and the cyphenothrin were charged tothe vessel and all components were mixed until homogenous solution wasformed (about 1 hour).

Example 2 Method of Making a Fipronil/S-Methoprene/Cyphenothrin Spot-OnPesticide Composition for Dogs

Ingredient Amount for 1000 L Fipronil tech. (As 100%) 98 kg (99.7 kg as98.3%) S-methoprene (As 100%) 88 kg (92.1 kg as 95.5%) Cyphenothrin (As100%) 50 kg (53 kg as 94.4%) Tocopherol Nicotinate 53 kg Diethyleneglycol monoethyl ether Up to 1000 L (about 702.2 kg)

The diethylene glycol monoethyl ether and the tocopherol nicotinate werecharged to a vessel and heated to a temperature of 50° C. (about 1hour). Once heated, the fipronil, the cyphenothrin, and the S-methoprenewere charged to the vessel and all components were mixed until ahomogenous solution was formed (about 1 hour).

Example 3 Method of Making a Fipronil/Etofenprox Spot-On PesticideComposition for Cats

Ingredient Amount for 1000 L Fipronil tech. (As 100%)  98 kg (99.7 kg as98.3%) Etofenprox (As 100%) 150 kg (158.9 kg as 94%) Diethylene glycolmonoethyl ether Up to 1000 L (about 741.4 kg )

The diethylene glycol monoethyl ether was charged to a vessel and heatedto a temperature of 50° C. (about 1 hour). Once heated, the fipronil andthe etofenprox were charged to the vessel and all components were mixeduntil a homogenous solution was formed (about 1 hour).

Example 4 Method of Making a Fipronil/Etofenprox/S-Methoprene Spot-OnPesticide Composition for Cats

Ingredient Amount for 1000 L Fipronil tech. (As 100%)  98 kg (99.7 kg as98.3%) Etofenprox (As 100%) 150 kg (158.9 kg as 94%) S-methoprene (As100%)  88 kg (123.6 kg as 95.5%) Diethylene glycol monoethyl ether Up to1000 L (about 617.8 kg)

The diethylene glycol monoethyl ether was charged to a vessel and heatedto a temperature of 50° C. (about 1 hour). Once heated, the fipronil,the etofenprox, and the S-methoprene were charged to the vessel and allcomponents were mixed until a homogenous solution was formed (about 1hour).

Example 5 Efficacy Evaluation of a Spot-on Composition ContainingFipronil and a Spot-On Composition Containing Fipronil and Cyphenothrinfor the Treatment of Fleas and Ticks on Dogs

A double blind, controlled study was performed to illustrate thedifference in kill rates for ticks and fleas between treatment with aspot-on composition containing fipronil and cyphenothrin prepared in asimilar manner to Example 1, and a composition comprising only fipronil.

A total of 18 dogs were randomized into one of three treatment groups.Group A, which was the control group, did not receive any treatment forfleas and ticks and was used as a point of comparison against the activetreatment regimens. Group B comprised an active treatment group thatreceived treatment with the fipronil/cyphenothrin spot-on compositioncombination of 8.38% (w/w) fipronil and 4.76% (w/w) cyphenothrin.Finally, Group C comprised an active treatment group that receivedtreatment with 8.8% (w/w) fipronil only. For all active treatments, aspot-on application was developed and applied to the dogs in a manner inaccordance with this invention. All dogs admitted into the experimentwere first deemed to be suffering from both flea and tick infestation.The experiment was designed such that all treatment groups wereadministered the appropriate treatment and then observed at one hour andat four hours after application. During the post-application observationperiods, the dogs were contacted with a piece of test paper in definedareas of the dog's body, for a defined period of time. The paper wasdesigned such that dead ticks and fleas would adhere to the paper, andthe number of dead ticks and fleas could be counted for comparison tothe Group A (control group), to determine the reduction in pestsfollowing treatment. In addition, the number of ticks and fleasremaining on the dog, within the observation areas, was also calculated.

The dogs of each group (Group A, Group B, and Group C) were infestedwith fleas and ticks on Day −1, and the two active treatment groups(Group B and Group C) subsequently received treatment on Day 0. Theprotocol was designed to test the efficacy of the two treatments inregard to subsequent infestation after an initial treatment with anactive spot-on composition. After the initial infestation, the dogs wereagain infested with fleas in four intervals on the first day of eachweek (Day 7, 14, 21, and 28) for a period of approximately 4 weeks, withmonitoring through Day 30. For each subsequent infestation, the dogs ofeach treatment group were monitored to determine the kill rates forfleas (Ctenophalides felis) and ticks (Rhipicephalus sanguineus) at onehour after infestation, four hours after infestation, one day (24 hours)after infestation, and two days (48 hours) after infestation. The valuesin Tables 1 and 2 represent the average percentage reduction in thenumber of ticks and fleas, respectively, present on the dogs in eachtreatment group after subsequent infestations, comparing treatmentresults for Group B (fipronil and cyphenothrin) with Group C (fipronil).

TABLE 1 Group Mean Efficacy Against Ticks Group Day Time (hrs) B C 0 1 31%  5% 4  69%  27% 24  87%  86% 48  96%  97% 7 1  95%  38% 4  94%  89%24 100% 100% 48 100% 100% 14 1  40%  5% 4  91%  85% 24 100%  99% 48 100%100% 21 1  98%  74% 4  99%  94% 24 100% 100% 48 100% 100% 28 1  96%  65%4 100%  93% 24 100% 100% 48 100% 100%

Table 1 provides information on the average tick kill rates for the twotreatment groups described above. The dogs in Group B, treated with acombination of 8.38% fipronil and 4.76% cyphenothrin, experiencedsignificantly greater average tick kill rates at one hour and four hoursafter treatment (Day 0) and after all re-infestation periods (Days 7,14, 21, and 28), compared to Group C (treatment with 8.8% fipronil).Thus, Table 1 illustrates that treatment with a combination of fiproniland cyphenothrin results in significantly greater average kill rates andspeed of kill for ticks at one hour and four hours after an initialtreatment and after subsequent re-infestations. Table 1 also illustratesthat treatment with a combination of fipronil and cyphenothrin providesprolonged efficacy for the treatment and prevention of tick infestationup to thirty days following application of the pesticidal composition.

TABLE 2 Group Mean Efficacy Against Fleas Group Day Time (hrs) B C 0 1 26%  9% 4  68%  35% 24 100% 100% 48 100% 100% 7 1  91%  44% 4  97%  93%24 100% 100% 48 100% 100% 14 1  85%  42% 4  46%  37% 24 100% 100% 48100% 100% 21 1  89%  72% 4  94%  92% 24 100% 100% 48 100% 100% 28 1  69% 12% 4  93%  76% 24 100% 100% 48 100% 100%

As is evident from Table 2, the dogs treated with a combination of 8.38%fipronil, and 4.76% cyphenothrin (Group B) experienced higher averageflea kill rates one hour and four hours after infestation for allsubsequent infestation periods (Days 7, 14, 21, and 28). At allre-infestation intervals, the dogs treated with fipronil andcyphenothrin (Group B) experienced greater average reductions in thenumber of fleas present at one and four hours post re-infestation. Thus,the results of this example illustrate that treatment with a combinationof fipronil and cyphenothrin results in significantly greater averagekill rates and speed of kill for fleas at one hour and four hours aftertreatment, and also after subsequent infestations, and providesprolonged treatment efficacy for up to thirty days following treatment.

The empirical data presented in Tables 1 and 2 illustrates thattreatment with a combination of fipronil and cyphenothrin results ingreater average kill rates and speed of kill for fleas and ticks onehour and four hours after treatment compared to treatment with fipronilalone. In addition, Tables 1 and 2 show that superior kill rates withthe combination of fipronil and cyphenothrin persisted after foursubsequent re-infestations of the host dogs with both fleas and ticks,up to thirty days after the initial application of the spot-oncomposition.

Example 6 Efficacy Evaluation of a Spot-On Composition ContainingFipronil and S-methoprene, and a Spot-On Composition ContainingFipronil, S-Methoprene, and Cyphenothrin for the Treatment of Fleas andTicks on Dogs

A double blind, randomized, single-centered, controlled study wasperformed to illustrate the efficacy of a fipronil and S-methoprenespot-on composition enhanced with cyphenothrin prepared in a similarmanner to Example 2, compared to fipronil and S-methoprene spot-oncompositions not enhanced with cyphenothrin in the treatment of ticksand fleas on dogs.

The study was conducted on thirteen groups of dogs, each groupconsisting of six dogs for a total of 78 dogs. Dogs were rankedaccording to body weight and randomly allocated to four body weightcategories: A (4 lbs to 22.9 lbs), B (23 lbs to 44.9 lbs), C (45 lbs to88.9 lbs) and D (25 lbs to 35.9 lbs). Control dogs (Group 1) wereselected to be representative of low, medium, and high body weights andhad acceptable numbers of fleas and ticks without being biased towardhigh parasite numbers. Within each weight category (A, B, C, D) the dogswere randomly allocated to groups 2, 3, or 4, thus forming three groups(2, 3, 4) within each weight category (A, B, C, D). Three spot-onformulations were topically administered, one to each group of dogs (2,3, and 4) as shown in Table 3. The fipronil and S-methoprene spot-onproducts (one reformulated for the test and one commercially available)each were comprised of 9.8% fipronil and 8.8% S-methoprene. Thefipronil, S-methoprene, and cyphenothrin spot-on product was comprisedof 9.8% fipronil, 8.8% S-methoprene, and 5.0% cyphenothrin. Dose volumesgiven to the treated dogs conformed to conventional dosages (i.e. 0.67mL to dogs weighing less than 23 lbs, 1.67 mL to dogs weighing between23 lbs and 44.9 lbs, and 2.68 mL to dogs weighing between 45 lbs and88.9 lbs). The fourth subgroups of dogs (D) were treated with the testsubstances at a volume dose rate of 0.67 mL/kg which provides thecalculated minimum active ingredients' dose rates that would be appliedto dogs at the upper limit in the group weighing from 89 to 132 lbs.

TABLE 3 Grouping and Treatment Group Weight Treatment 1  4 lb-60 lb None2A  4 lb-22.9 lb Reformulated fipronil/S-methoprene 2B 23 lb-44.9 lbReformulated fipronil/S-methoprene 2C 45 lb-88.9 lb Reformulatedfipronil/S-methoprene 2D 25 lb-35.9 lb Reformulatedfipronil/S-methoprene 3A  4 lb-22.9 lbfipronil/S-methoprene/cyphenothrin 3B 23 lb-44.9 lbfipronil/S-methoprene/cyphenothrin 3C 45 lb-88.9 lbfipronil/S-methoprene/cyphenothrin 3D 25 lb-35.9 lbfipronil/S-methoprene/cyphenothrin 4A  4 lb-22.9 lb Retailfipronil/S-methoprene 4B 23 lb-44.9 lb Retail fipronil/S-methoprene 4C45 lb-88.9 lb Retail fipronil/S-methoprene 4D 25 lb-35.9 lb Retailfipronil/S-methoprene

The three active treatments were applied to the respective groups onceat the beginning of the study (Day 0). The dogs were infested with fleas(Ctenophalides felis) and ticks (Rhipicephalus sanguineus, Dermacentorvariabilis, and Haemaphysalis elliptica) in five intervals (Day −1, 7,14, 21, and 28) for a period of approximately 4 weeks, with monitoringthrough Day 30 after treatment. For each subsequent infestation, thedogs of each treatment group were monitored to determine the kill ratesfor ticks at one hour after infestation, four hours after infestation,one day (24 hours) after infestation, and two days (48 hours) afterinfestation. Ticks were found by direct observation following parting ofthe hair coat and palpation in each area of the animal being examined(i.e. outside hind legs including feet, tail and anal areas, lateralarea not including shoulders, abdominal area from chest to inside hindlegs, fore legs and shoulders including feet, all neck and head areas,and dorsal strip from shoulder blades to base of tail). The efficacies(%) of the respective treatments on ticks at 24 and 48 hours aftertreatment/re-infestation and at 1 and 4 hours aftertreatment/re-infestation are shown in Tables 4 and 5.

Flea counts were made only at 48 hours after treatment (i.e. Day 2) andafter each re-infestation (i.e. Days 9, 16, 23, and 30). A fine-toothedcomb was used to recover fleas present in the animal's fur. The methodof combing was by several strokes of the comb in each area of the animalbeing examined (i.e. outside hind legs including feet, tail and analareas, lateral area not including shoulders, abdominal area from chestto inside hind legs, fore legs and shoulders including feet, all neckand head areas, and dorsal strip from shoulder blades to base of tail).Each stroke of the comb was made in the same direction following thepattern of the hair coat. Movement from one part of the animal's fur tothe next was via strokes overlapping each other so that no area of furwas missed. The efficacies of the treatments on fleas are shown in Table6.

TABLE 4 Group Mean Efficacy Against Ticks at 24 & 48 Hours AfterTreatment/Re- infestation Time Group Day (hrs) 2A 2B 2C 2D 3A 3B 3C 3D4A 4B 4C 4D 0 24 61% 78% 96% 0% 30% 74% 91% 39% 74% 52% 78% 70% (DV) 4876% 82% 100% 76% 80% 94% 100% 82% 88% 94% 94% 94% 0 24 73% 68% 74% 56%83% 57% 92% 71% 61% 61% 86% 73% (RS) 48 73% 79% 97% 67% 90% 81% 100% 84%94% 67% 90% 97% 7 24 94% 96% 97% 97% 100% 99% 100% 98% 98% 98% 97% 89%(DV) 48 98% 100% 100% 100% 100% 100% 100% 99% 100% 100% 100% 100% 7 2496% 96% 97% 95% 100% 100% 100% 95% 100% 92% 92% 92% (RS) 48 99% 100%100% 100% 100% 100% 100% 100% 100% 100% 95% 95% 14  24 91% 97% 97% 85%49% 100% 77% 86% 91% 88% 81% 76% (HE) 48 94% 100% 98% 91% 80% 98% 100%95% 98% 98% 94% 90% 21  24 92% 90% 96% 72% 69% 86% 92% 92% 55% 56% 93%49% (DV) 48 100% 100% 100% 97% 98% 100% 100% 95% 95% 86% 100% 80% 21  2495% 87% 97% 80% 99% 99% 99% 85% 79% 83% 85% 66% (RS) 48 99% 98% 98% 86%100% 99% 100% 95% 97% 90% 99% 79% 28  24 70% 94% 72% 36% 75% 18% 87% 70%34% 15% 18% 1% (DV) 48 99% 99% 100% 65% 93% 68% 99% 90% 92% 64% 93% 71%28  24 59% 90% 97% 66% 89% 84% 94% 56% 31% 54% 70% 31% (RS) 48 96% 99%98% 90% 95% 94% 98% 94% 93% 85% 94% 69% DV = infested with D.variabilis; RS = infested with R. sanguineus; HE = infested with H.elliptica

As illustrated in Table 4, efficacy against existing ticks (Day −1infestation) was irregular for all test substances at 24 and 48 hoursafter initial treatment. Overall, greater average tick kill rates forthe treatment enhanced with cyphenothrin (Groups 3A, 3B, 3C, and 3D)were observed in all dog weight classes against both species of ticksrecorded 24 hours following infestations. The effectiveness of thefipronil treatment enhanced with cyphenothrin against ticks 48 hoursafter treatment was >90% or just marginally below 90% for the differentdog weight classes treated according to dose ranges. Table 4 alsoillustrates that treatment with a combination of fipronil/IGR andcyphenothrin provides prolonged efficacy for the treatment andprevention of tick infestation up to thirty days following applicationof the composition.

TABLE 5 Group Mean Immediate Efficacy Against Ticks at 1 & 4 Hours AfterTreatment/Re-infestation Treatment Time Group Day (hrs) 2A 2B 2C 2D 3A3B 3C 3D 4A 4B 4C 4D  0 (DV) 1 0% 0% 14% 0% 0% 0% 0% 0% 0% 0% 25% 0% 40% 17% 39% 0% 0% 0% 33% 0% 0% 0% 56% 0%  0 (RS) 1 8% 29% 0% 0% 16% 0%21% 0% 0% 0% 14% 12% 4 51% 53% 34% 33% 53% 40% 53% 38% 35% 36% 53% 66% 7 (DV) 1 34% 50% 47% 50% 63% 73% 85% 63% 0% 45% 46% 23% 4 53% 62% 60%51% 86% 82% 85% 59% 50% 32% 65% 39%  7 (RS) 1 28% 57% 48% 30% 60% 78%83% 66% 50% 59% 41% 42% 4 75% 79% 92% 74% 86% 94% 92% 77% 81% 77% 80%60% 14 (HE) 1 31% 0% 0% 19% 0% 8% 21% 0% 0% 24% 57% 19% 4 31% 20% 51%14% 0% 28% 17% 0% 7% 23% 40% 23% 21 (DV) 1 2% 16% 21% 0% 43% 15% 48% 36%0% 2% 2% 0% 4 17% 26% 25% 0% 33% 26% 47% 38% 0% 0% 44% 0% 21 (RS) 1 0%15% 18% 28% 55% 64% 74% 73% 14% 19% 36% 0% 4 45% 28% 67% 46% 63% 86% 94%60% 39% 39% 40% 9% 28 (DV) 1 0% 0% 0% 0% 0% 0% 0% 29% 0% 0% 0% 0% 4 0%0% 24% 0% 0% 0% 0% 31% 0% 0% 1% 0% 28 (RS) 1 8% 38% 0% 40% 8% 22% 62%32% 0% 4% 0% 22% 4 37% 40% 39% 64% 61% 63% 67% 41% 12% 24% 13% 24% DV =infested with D. variabilis; RS = infested with R. sanguineus; HE =infested with H. elliptica

Table 5 illustrates that the enhanced spot-on composition consistentlyprovided better immediate efficacy against new ticks, especially whencompared to the compositions lacking cyphenothrin (Groups 2 and 4).Efficacy in killing/repelling ticks at 1 and 4 hours afterre-infestation was observed for both species of ticks on Days 7 and 21and for R. sanguineus on Day 28. Thus, Table 5 illustrates thattreatment with a combination of fipronil and cyphenothrin results insignificantly greater average kill rates and speed of kill for ticks atone hour and four hours after an initial treatment and after subsequentre-infestations.

TABLE 6 Group Mean Efficacy Against Fleas at 48 Hours afterTreatment/Re-infestation Treatment Group Day 2A 2B 2C 2D 3A 3B 3C 3D 4A4B 4C 4D 2 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 99% 9100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 16 100% 100%100% 100% 100% 100% 100% 100% 100% 100% 100% 100% 23 100% 100% 100% 100%100% 100% 100% 100% 100% 100% 100% 100% 30 100% 100% 100% 100% 100% 100%100% 100% 100% 97% 100% 98%

Table 6 illustrates that the efficacy of the composition enhanced withcyphenothrin was at 100% for all dog weights throughout the study (i.e.up to Day 30), whereas the efficacy of the retail fipronil/IGRcomposition failed to remain at 100% efficacy for mid-weight range dogs.

In summary, efficacy of the enhanced formulation against fleas, measuredat 48 hours after treatment and after re-infestation was 100%. Thecyphenothrin-enhanced formulation was often more efficacious compared tothe commercially marketed fipronil/IGR formulation not containingcyphenothrin. Residual efficacy against new tick infestations over the30 days after treatment was well above 90% in all of the weight groups,but was much less than 90% in half of the weight groups for thecommercial product in the final week of the study. Enhancement of theformulation with 5% cyphenothrin improved immediate efficacy against newticks (D. variabilis and R. sanguineus) on the 7^(th) and 21^(st) daysand for R. sanguineus on the 28^(th) day. Comparing the enhancedformulation with the two fipronil-only test substances showed that theenhanced test substance provided statistically greater repellent actionat 1 and 4 hours after re-infestation.

Example 7 Efficacy Evaluation of a Spot-On Composition ContainingFipronil/S-Methoprene and a Spot-On Composition ContainingFipronil/S-Methoprene/Etofenprox for the Treatment of Fleas and Ticks onCats

A double blind, randomized, single-centered, controlled study wasperformed to illustrate the efficacy of a fipronil/IGR spot-oncomposition enhanced with etofenprox compared to fipronil/IGR spot-oncompositions not enhanced with etofenprox in the treatment of ticks andfleas on cats. An experimental spot-on formulation comprising 9.8%fipronil and 11.8% S-methoprene was enhanced by adding 15% etofenprox.This enhanced composition, along with two fipronil/S-methopreneformulations not enhanced with etofenprox, were evaluated for efficacyagainst pre-existing fleas and ticks.

The study was conducted on seven groups of cats, each group consistingof six cats for a total of 42 cats. The cats were randomly allocated tothe seven groups. Etofenprox technical active (97% a.i.) was added atthe rate of 15% to a basic fipronil-methoprene formulation (9.8%fipronil, 11.8% S-methoprene) to provide an enhanced formulation. Thetwo fipronil-methoprene formulations not containing etofenprox werecomprised of 9.8% and 11.8% S-methoprene. One fipronil/methopreneformulation was reformulated for the test and one was commerciallyavailable, which served as the positive control for the study. Theenhanced formulation was applied at a 0.50 mL unit dose to a group ofsix treated cats (Group 2A). The unenhanced formulations were applied ata 0.50 mL unit dose to two groups each of six cats (Groups 3A and 4A).Three additional groups, each of six cats, were treated by applying thetest substances at the minimum dose rate, equivalent to treating catsweighing 20 lb with dose volumes of 0.5 mL (approximately 0.055 mL/kg).The enhanced formulation was administered at this dosage to Group 2B andthe unenhanced formulations were administered at this dosage to Groups3B and 4B. The weight specifications for all treated cats were 10 lb+/−5lb. A seventh group of six cats (Group 1) served as untreated controls.Table 7 sets forth the grouping, treatment and dosage of the cats in thestudy.

TABLE 7 Grouping, Treatment, and Dosage Group Weight Dosage Treatment 15 lb-15 lb None None 2A 5 lb-15 lb  0.5 mL/catfipronil/S-methoprene/etofenprox 2B 5 lb-15 lb 0.055 mL/kgfipronil/S-methoprene/etofenprox 3A 5 lb-15 lb  0.5 mL/cat Reformulatedfipronil/S-methoprene 3B 5 lb-15 lb 0.055 mL/kg Reformulatedfipronil/S-methoprene 4A 5 lb-15 lb  0.5 mL/cat Retailfipronil/S-methoprene 4B 5 lb-15 lb 0.055 mL/kg Retailfipronil/S-methoprene

The cats were infested with 100 fleas (Ctenophalides felis) on Days −1,7, 14, 21, and 28 and 50 ticks (Rhipicephalus turanicus on Day 7,Dermacentor variabilis on Days −1, 21, and 28), and Haemaphysaliselliptica on Day 14) for a period of approximately 4 weeks, withmonitoring through Day 30 after treatment. Tick counts, withoutdisturbing or removing ticks, were conducted at one hour and four hoursafter application of the test substances on the day of treatment (Day 0)and similarly after re-infestation on Days 8, 15, 22, and 28, in orderto determine the speed of kill as evidence of repellency. Tick countswere also made at 24 and 48 hours after treatment and after eachre-infestation. Ticks were found by direct observation following partingof the hair coat and palpation in each area of the animal being examined(i.e. outside hind legs including feet, tail and anal areas, lateralarea not including shoulders, abdominal area from chest to inside hindlegs, fore legs and shoulders including feet, all neck and head areas,and dorsal strip from shoulder blades to base of tail). The efficacies(%) of the respective treatments on ticks at 24 hours and 48 hours aftertreatment and after re-infestation are shown in Table 8. The efficacies(%) of the respective treatments on ticks at 1 hour and 4 hours aftertreatment and after re-infestation are shown in Table 9.

TABLE 8 Group Mean Efficacy Against Ticks at 24 & 48 Hours AfterTreatment/Re-infestation Treatment Time Group Day (hrs) 2A 2B 3A 3B 4A4B  0 (DV) 24 75% 77% 94% 50% 87% 65% 48 100% 86% 100%  58% 100% 51%  7(RT) 24 98% 77% 100%  87% 100% 55% 48 100% 98% 100%  100% 98% 100% 14(HE) 24 62% 77%  90%* 58% 73% 55% 48 99% 88% 99% 89% 99% 89% 21 (DV) 2491% 77% 92% 97% 91% 84% 48 99% 89% 99% 98% 92% 91% 28 (DV) 24 90% 85% 90%* 68% 91% 36% 48 98% 91% 99% 72% 94% 46% DV = infested with D.variabilis; RT = infested with R. turanicus; HE = infested with H.elliptica Bold type indicates meets/exceeds 90% (unless roundingincreases to 90%*)

As shown in Table 8, all three test substances at full label dose (0.5mL) performed similarly by attaining/exceeding 90% efficacy forkilling/repelling ticks at 48 hours after treatment and re-infestation.Occasionally, adequate efficacy (=/>90%) against ticks was shown also at24 hours after treatment or after re-infestation, almost exclusively forthe groups that receive the full label dose rate. Groups treated at theminimum dose rate (Groups 2B, 3B, and 4B) attained 90% efficacy ormarginally below 90% efficacy (i.e. at least between 85%-89%) againstfleas at 48 hours, except for the positive control group (Group 4B) inthe fourth week.

TABLE 9 Group Mean Efficacy Against Ticks at 1 and 4 Hours afterTreatment/Re-infestation Treatment Time Group 10 Day (hrs) 2A 2B 3A 3B4A 4B  0 (DV) 1 0% 2% 52% 7% 21% 24% 4 15% 12% 51% 57% 34% 49%  7 (RT) 146% 2% 30% 14% 0% 0% 4 59% 12% 35% 29% 0% 6% 14 (HE) 1 0% 27% 0% 0% 0%0% 4 0% 63% 5% 4% 0% 0% 21 (DV) 1 56% 27% 69% 72% 50% 35% 4 91% 63% 89%98% 85% 86% 28 (DV) 1 57% 47% 47% 0% 65% 0% 4 89% 86% 81% 66% 92% 37% DV= infested with D. variabilis; RT = infested with R. turanicus; HE =infested with H. elliptica Bold type indicates meets/exceeds 90%

Table 9 illustrates that efficacy in killing/repelling ticks at 1 hourand 4 hours after treatment and after re-infestation did not showimprovement in immediate efficacy attributable to the etofenproxenhancement of the basic fipronil-methoprene formulation. On only threeoccasions (at 4 hour counts) was adequate (=/>90%) immediate efficacyshown against ticks and these three events did not appear to beconsistently related to formulation or to dose rate.

TABLE 10 Group Mean Efficacy Against Fleas at 48 Hours AfterTreatment/Re-infestation Treatment Group Day 2A 2B 3A 3B 4A 4B 2 99%100% 100% 99% 99% 98% 9 100% 100% 100% 100% 100% 100% 16 100% 100% 100%100% 100% 100% 23 100% 100% 100% 100% 100% 98% 30 97% 93% 100% 99% 100%85% Bold type indicates meets/exceeds 90%

Table 10 illustrates that the three treatments administered to cats at aminimal dose of 0.055 mL/kg all had a greater than 90% immediateefficacies against fleas and persistent efficacies >90% for four weeks(Groups 2B and 3B) and three weeks (Group 4B). Table 10 further showsthat the three treatments administered to cats at a dose of 0.50 mL/catall had immediate (Day 2) and four week persistent efficacies of >90%against fleas.

In summary, the only test substance that provided 90% or better efficacyagainst fleas and ticks at both full and minimum dose rate at the end ofthe study was the enhanced fipronil-methoprene (Group 2) and thepositive control test substances (Group 4), when administered at fulllabel dose rate only (Groups 2A and 4A). Both also provided adequateefficacy against fleas and ticks in the fourth week of the study.

Example 8 Testing of a Fipronil and S-Methoprene Composition Against theEnhanced Fipronil, S-Methoprene, and Cyphenothrin Composition

A double blind, controlled study was performed to illustrate thedifference in kill rates for ticks and fleas between treatment with aspot-on composition containing fipronil and S-methoprene and treatmentwith an enhanced spot-on composition comprising fipronil, S-methoprene,and cyphenothrin prepared in a similar manner to Example 2.

A total of 18 dogs were randomized into three treatment groups. Group Awas the control group and did not receive treatment. Group B comprisedan active treatment group that received treatment with the enhancedcombination of 9.8% fipronil, 8.8% S-methoprene, and 5% cyphenothrin.Group C comprised an active treatment group that received treatment withthe combination of 9.8% fipronil and 8.8% S-methoprene. For alltreatments, a spot-on application was developed and applied to the dogsin a manner in accordance with this invention. All dogs admitted intothe experiment were first deemed to be suffering from both flea and tickinfestation. The experiment was designed such that all treatment groupswere administered the appropriate treatment and then observed one hourand four hours after application. During the post-applicationobservation periods, the dogs were contacted with a piece of test paperin defined areas of the dog's body, for a defined period of time. Thepaper was designed such that dead ticks and fleas would adhere to thepaper, and the number of dead ticks and fleas could be counted forcomparison to the control group (Group A) to determine the reduction inpests due to treatment. In addition, the number of ticks and fleasremaining on the dog within the observation area was also calculated.The results of the experiment are shown in Table 11.

TABLE 11 Kill Data for Treatment of Ticks and Fleas +1 Hour +4 HoursDead On Dead On Total Dead On Dog Paper On Dog Paper on Paper Run # CageDog # Group Ticks Fleas Ticks Fleas Ticks Fleas Ticks Fleas Ticks Fleas30 110 468 A 19 26 0 1 18 20 0 0 0 1 32 112 411 A 34 38 0 0 25 34 0 0 00 34 114 357 A 32 56 0 1 36 52 0 2 0 3 36 116 356 A 21 28 0 0 22 23 0 00 0 38 118 457 A 31 39 0 1 30 35 0 1 0 2 40 120 424 A 41 33 0 0 35 30 02 0 2 Average 29.7 36.7 0.0 0.5 27.7 32.3 0.0 0.8 0.0 1.3 44 IS1 471 B 98 18 25 6 1 10 18 28 43 46 IS2 430 B 0 3 48 59 0 0 1 6 49 65 48 11 229 B1 0 49 42 0 0 3 12 52 54 50 50 301 B 6 13 36 36 0 6 7 24 43 60 52 52 429B 2 10 46 58 0 1 2 14 48 72 54 54 412 B 4 20 43 39 0 0 4 24 47 63Average 3.7 9.0 40.0 43.2 1.0 1.3 4.5 16.3 44.5 59.5 % Reduction 87.6%75.5% 96.4% 95.9% 58 58 420 C 24 18 8 6 0 5 20 32 28 38 60 60 470 C 3447 4 13 1 10 18 34 22 47 62 62 222 C 25 34 7 14 1 5 25 33 32 47 64 64449 C 28 40 3 4 3 21 27 35 30 39 66 66 464 C 27 33 7 5 2 2 30 53 37 5868 68 463 C 25 21 4 6 2 3 25 47 29 53 Average 27.2 32.2 5.5 8.0 1.5 7.724.2 39.0 29.7 47.0 % Reduction 8.4% 12.3% 94.6% 76.3%

As is evident from Table 11, Group B (treatment including cyphenothrin)experienced a greater decrease in the number of ticks and fleas killedwithin the first hour of treatment compared to Group C (treatmentwithout cyphenothrin). Using Group A as an average baseline for thenumber of ticks and fleas present on an infested dog, the dogs in GroupB experienced a 87.6% and 75.5% decrease in the average number of ticksand fleas present on the dog's skin, respectively, after one hour.Alternatively, the dogs in Group C only experienced an 8.4% and 12.3%decrease in the average number of ticks and fleas present on the dog'sskin, respectively, after one hour.

Furthermore, at the four-hour interval, dogs in Group B experienced anaverage reduction in the number of ticks and fleas remaining on the skinof the animal of 96.4% and 95.9%, respectively. The dogs in Group Cexperienced an average reduction of 94.6% and 76.3%. Thus, the averagereduction in ticks on the animals was similar, but the average reductionin fleas on the dog's skin was significantly higher (95.9% in Group Bvs. 76.3% in Group C). Finally, the total number of dead ticks and fleasdead on the paper was higher for Group B, compared to Group C. TreatmentGroup B experienced a total kill rate, on average, of 44.5 ticks and59.5 fleas over the course of the four-hour interval, compared to atotal average kill rate of 29.7 ticks and 49.0 fleas for treatment GroupC. As such, treatment with a combination of fipronil, S-methoprene, andcyphenothrin resulted in a significantly higher average kill rate afterone hour, and maintained a greater average kill rate for both ticks andfleas, four hours after treatment.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by thefollowing claims.

What is claimed is:
 1. A method of killing insect and pest pupae andadults on an animal, which method comprises administering a localizedcutaneous application between the shoulders of the animal, a spot-oncomposition comprising 8% to 11% (w/w) fipronil, 3% to 16% (w/w)cyphenothrin, and 60% to 80% (w/w) organic solvent.
 2. The method ofclaim 1, wherein the spot-on composition comprises 4% to 6% (w/w) ofcyphenothrin.
 3. The method of claim 1, wherein the animal is a mammal.4. The method of claim 3, wherein the mammal comprises a dog or a cat.5. The method of claim 1, wherein the spot-on composition additionallycomprises an insect growth regulator.
 6. The method of claim 5, whereinthe insect growth regulator is present in the composition at aconcentration of between 2% to 15% (w/w).
 7. The method of claim 1,wherein the spot-on composition additionally comprises 4% to 6% (w/w) ofan antioxidant.
 8. The method of claim 1, wherein the compositionprovides at least 90% efficacy against insect and pest pupae for aperiod of at least 30 days following administration of the compositionto the animal.
 9. The method of claim 1, wherein the composition isadministered to the animal in a volume sufficient to deliver to theanimal a dosage of cyphenothrin ranging from about 0.1 mg/kg to about 40mg/kg.
 10. A method of killing insect and pest pupae and adults on ananimal, which method comprises administering a localized cutaneousapplication between the shoulders of the animal, a spot-on compositioncomprising: a. 8% to 11% (w/w) fipronil; b. 4% to 6% (w/w) cyphenothrin;c. 70% to 80% (w/w) organic solvent; and d. 4% to 6% (w/w) antioxidant.11. The method of claim 10, wherein the organic solvent is diethyleneglycol monoethyl ether.
 12. The method of claim 10, wherein theantioxidant is tocopherol nicotinate.
 13. The method of claim 12,wherein the organic solvent is diethylene glycol monoethyl ether. 14.The method of claim 12, wherein the composition further comprises 2% to15% (w/w) insect growth regulator.
 15. The method of claim 14, whereinthe insect growth regulator comprises pyriproxyfen or S-methoprene. 16.A method of killing insect and pest pupae and adults on an animal, whichmethod comprises administering a localized cutaneous application betweenthe shoulders of the animal, a spot-on composition comprising: a. 8% to11% (w/w) fipronil; b. 4% to 6% (w/w) cyphenothrin; c. 8% to 12% (w/w)S-methoprene; d. 70% to 80% (w/w) organic solvent; and e. 4% to 6% (w/w)antioxidant.
 17. The method of claim 16 wherein the animal is a mammal.18. The method of claim 17 wherein the mammal comprises a dog or a cat.19. A method of killing insect and pest pupae and adults on an animal,which method comprises administering a localized cutaneous applicationbetween the shoulders of the animal, a spot-on composition comprising:a. 8% to 11% (w/w) fipronil; b. 4% to 6% (w/w) cyphenothrin; c. 3% to 5%(w/w) pyriproxyfen; d. 70% to 80% (w/w) organic solvent; and e. 4% to 6%(w/w) antioxidant.
 20. A method of killing insect and pest pupae andadults on an animal, which method comprises administering a localizedcutaneous application between the shoulders of the animal, a spot-oncomposition comprising: a. 8% to 11% (w/w) fipronil; b. 4% to 6% (w/w)cyphenothrin; and c. 70% to 80% (w/w) diethylene glycol monoethyl ether.21. The method of claim 20, wherein the composition further comprises 3%to 5% (w/w) pyriproxyfen.
 22. The method of claim 20, wherein thecomposition further comprises 8% to 12% (w/w) S-methoprene.